Ana Oleaga

Characterization of widespread canine leishmaniasis among wild carnivores from Spain

Visceral Leishmaniasis (VL) is an emerging zoonotic parasitic disease caused by Leishmania infantum in Mediterranean countries, with sand flies (Phlebotomus spp.) as vectors and dogs as the main domestic reservoir. The role of wild carnivores in the epidemiology of leishmaniasis is still controversial. In order to determine the prevalence of natural infection with L. infantum in wild carnivores from Spain, we analyzed 217 samples by PCR and western blotting and used restriction fragment length polymorphism (RFLP) to compare the patterns present in wild carnivores with those of domestic dogs from the same areas. DNA of the parasite was detected in spleen or blood samples from 35 (16.12%) analyzed wild carnivores, including 8 of 39 (20.5%) wolves (Canis lupus), 23 of 162 (14.1%) foxes (Vulpes vulpes), 2 of 7 (28.6%) Egyptian mongooses (Herpestes ichneumon), 1 of 4 genets (Geneta geneta), and 1 of 4 Iberian lynxes (Lynx pardinus). No significant sex or age differences in prevalence were observed in wolves and foxes (P>0.05), but there was a significant difference among regions in foxes (P<0.05). A total of 12 PCR-RFLP patterns were found in foxes, 6 in wolves, 4 in dogs, 2 in Egyptian mongooses and 1 in lynx and genet. RFLP patterns differed between dogs and foxes in the two areas where they could be compared. This is the first study of canine leishmaniasis in wild canids and other carnivores from different regions of Spain by PCR. The prevalence of infection indicates the existence of natural infection in apparently healthy wild carnivore populations, and our results are suggestive of a sylvatic cycle independent of dogs.

Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins

The current study was designed to test the immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in sheep subsequently infected with the fluke. Thirty lambs were divided into six groups according to various patterns of immunisation and times of infection and necropsy. The antigens were emulsified in Freunds adjuvant. Levels of specific anti-nFh12 and anti-rFh15 antibodies rose rapidly by 2 weeks after the first immunisation and were always significantly higher in immunised-infected sheep than in control-infected sheep. On completion of the trial there was no difference in fluke burden between groups vaccinated with either of the antigens and non-immunised controls. However, worm size and faecal egg counts were significantly diminished in the sheep vaccinated with either of the antigens, suggesting an anti-fecundity effect. This is the first report of experimental vaccination of sheep against F. hepatica with purified native and recombinant antigens related to fatty acid binding proteins.

Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study

BACKGROUND Schistosomiasis is a snail-borne parasitic disease endemic in several tropical and subtropical countries. However, in the summer of 2013, an unexpected outbreak of urogenital schistosomiasis occurred in Corsica, with more than 120 local people or tourists infected. We used a multidisciplinary approach to investigate the epidemiology of urogenital schistosomiasis in Corsica, aiming to elucidate the origin of the outbreak. METHODS We did parasitological and malacological surveys at nine potential sites of infection. With the snails found, we carried out snail-parasite compatibility experiments by exposing snails to schistosome larvae recovered from the urine of a locally infected Corsican patient. Genetic analysis of both mitochondrial (cox1) and nuclear (internal transcribed spacer) DNA data from the Schistosoma eggs or miracidia recovered from the infected patients was conducted to elucidate the epidemiology of this outbreak. FINDINGS We identified two main infection foci along the Cavu River, with many Bulinus truncatus snails found in both locations. Of the 3544 snails recovered across all sites, none were naturally infected, but laboratory-based experimental infections confirmed their compatibility with the schistosomes isolated from patients. Molecular characterisation of 73 eggs or miracidia isolated from 12 patients showed infection with Schistosoma haematobium, S haematobium-Schistosoma bovis hybrids, and S bovis. Further sequence data analysis also showed that the Corsican schistosomes were closely related to those from Senegal in west Africa. INTERPRETATION The freshwater swimming pools of the Cavu River harbour many B truncatus snails, which are capable of transmitting S haematobium-group schistosomes. Our molecular data suggest that the parasites were imported into Corsica by individuals infected in west Africa, specifically Senegal. Hybridisation between S haematobium and the cattle schistosome S bovis had a putative role in this outbreak, showing how easily and rapidly urogenital schistosomiasis can be introduced and spread into novel areas where Bulinus snails are endemic, and how hybridisation could increase the colonisation potential of schistosomes. Furthermore our results show the potential risk of schistosomiasis outbreaks in other European areas, warranting close monitoring and surveillance of all potential transmission foci. FUNDING WHO, ANSES, RICET, and the Ministry of Health and Consumption.

African swine fever virus transmission cycles in Central Europe: Evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen

BackgroundAfrican swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format.ResultsOut of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely.ConclusionIn conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms.

Schistosoma bovis, a parasite of ruminants, can live for years in the bloodstream in spite of the immune response of its host. The parasite tegument covers the entire surface of the worm and plays a key role in the host-parasite relationship. The parasite molecules involved in host immune response evasion mechanisms must be expressed on the tegument surface and are potential targets for immune or drug intervention. The purpose of the present work was to identify the tegumental proteomes of male and female S. bovis worms, in particular the proteins expressed on the outermost layers of the tegument structure. Adult worms of each sex were treated separately with trypsin in order to digest their tegumental proteins, after which the peptides released were analysed by LC-MS/MS for identification. This experimental approach afforded valuable information about the protein composition of the tegument of adult S. bovis worms. A range of tegumental proteins was identified, most of which had not been identified previously in this species. Although an absolute purification of the proteins expressed on the outermost layers of the tegument structure was not achieved, it is likely that present among the proteins identified are some of the molecules most closely associated with the tegument surface. Our study also suggests that there may be differences in the protein composition of the tegument of male and female schistosomes. Finally, the presence of actin and GAPDH on the surface of male and female worms and the presence of enolase exclusively on the surface of male worms were verified by confocal microscopy.

Cloning and characterization of a plasminogen-binding surface-associated enolase from Schistosoma bovis.

Schistosoma bovis is a ruminant parasite able to survive prolonged periods in the vasculature of its host without either being cleared by the host defensive systems or inducing thrombotic or coagulation disturbances. This suggests that the parasite modulates both the immune and haemostatic host responses. Previous studies have shown that host plasminogen binds to the surface of S. bovis adult worms, and that a tegument extract from S. bovis fixes and activates host plasminogen, generating plasmin, which in turn could both inhibit blood clotting and dissolve clots. Enolase has been identified among the tegumental proteins that bind plasminogen. The aim of the present study is to determine the physiological role of the enolase found in the tegument of S. bovis adult worms as regards plasminogen-binding and activation, and to confirm its surface exposure on the parasite. The study included the cloning and sequencing of S. bovis enolase cDNA, collection of the corresponding recombinant protein and evaluation of its plasminogen-binding and activation activity, and an exploration of the expression and localization of native enolase in adult worms and lung schistosomulae. Here we show that S. bovis male adult worms express enolase on their tegumental surface and that this protein binds host plasminogen and increases its activation in the presence of host tissue plasminogen activator (t-PA). This suggests that the surface-associated enolase found here is a physiological receptor of plasminogen that plays a role in the activation of the host fibrinolytic system, most probably to avoid blood clot formation on the worms surface.

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freunds adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.

An insight into the proteome of the saliva of the argasid tick Ornithodoros moubata reveals important differences in saliva protein composition between the sexes

Tick saliva contains pharmacologically active molecules that allow these parasites to obtain a blood meal from the host and facilitate host infection by tick-borne pathogens. Recent transcriptomic and proteomic analyses of the salivary glands of several tick species have provided data sets that are invaluable for a better understanding of tick sialomes and tick-host-pathogen relationships. Here we performed a proteomic study of the saliva from the argasid tick Ornithodoros moubata. Saliva samples from female and male specimens were analyzed separately by LC-MS/MS before and after their equalization to facilitate the identification of the less abundant proteins. We report the array of 193 proteins identified in the saliva of O. moubata showing: (i) the broad and complex composition of the saliva of this tick, in good agreement with the complexity of the argasid and ixodid sialomes described previously; (ii) a notable difference in the saliva proteomes of females and males, since only 10 of the proteins identified appeared to be shared by both sexes; and (iii) the presence in the salivary fluid of a wide range of proteins known to be housekeeping/intracellular, which could be secreted in unconventional ways, including exosome secretion.

Vaccination of mice against Schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freunds adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.

Sarcoptic mange in red deer from Spain: improved surveillance or disease emergence?

Concern about emerging diseases has risen in recent years, and multihost situations have become increasingly relevant for wildlife management and conservation. We present data on Asturias, northern Spain, where 80 mangy red deer (Cervus elaphus) have been found since the beginning of the epizootic in chamois (Rupicapra pyrenaica parva) in 1993. We combine field and necropsy data with the results of a serosurvey using an in-house ELISA test to evaluate if deer mange due to Sarcoptes scabiei is an emerging disease in this area. The mean number of deer mange cases per year was 5, with a maximum of 16. No significant relationship was detected between monthly temperatures, rainfall or number of days with snow cover and the annual number of sarcoptic mange cases in red deer. Only 4 mangy red deer (5%) were detected outside the limits of scabietic chamois distribution during the same year, and all were less than 2500 m away from that limit. The longest distance reported between two consecutive mangy deer locations was 18 km. Mange cases were significantly more frequent in stags than in hinds and in adults than in juvenile deer. The time of the first mange detection in chamois in each sector, year with minimum number of chamois recorded, year with maximum chamois population decline rate and chamois density offered no significant correlation with red deer mange cases appearance moment and frequency. In the mange affected area, ELISA testing of 327 blood samples from hunter-harvested deer without obvious mange-compatible lesions revealed only 4 seropositive animals. All 83 sera from hunting preserves without clinical cases yielded negative ELISA results. According to these epidemiological data mange does not seem to threaten red deer populations in Asturias. However, continued monitoring of deer health and ELISA testing for sarcoptic mange is advisable.